MicroRNAs (miRNAs), ~22 nt long, single-stranded RNAs, guide protein complexes to block expression of mRNAs to which they bind by base pairing. The first microRNA was discovered in 1993; the second, in 2000. Currently, 3,229 microRNAs have been identified in plants, animals and viruses. As a class, miRNAs may rival transcription factors for their importance in orchestrating changes in gene expression. Our goal is to understand how miRNAs are made, assembled into functional complexes, and how these complexes regulate mRNA expression. We use Drosophila as a model system, because it offers powerful genetic and biochemical tools and because the miRNA pathway is closely conserved between flies and humans. What we learn in flies, we test in mammalian cell extracts and in cultured human and mouse cell lines. Our goal is to identify where these processes are conserved and where they diverge between flies and mammals, so as to understand the common logic of the miRNA pathway in animals and the unique features that have evolved in mammals. Pre-miRNAs, the immediate precursors of miRNAs, are -65 nt long RNA stem loop structures; the stems of pre-miRNA are imperfect, with G:U wobble pairs, mismatches, and internal loops interrupting a stem approximately three helical turns long. We will use quantitative biochemical and molecular tools to identify the proteins and protein complexes required to produce miRNA from pre-miRNAs, and to determine how these proteins enhance the accuracy and efficiency of pre-miRNA processing. Dicer, the enzyme that converts pre-miRNAs to miRNAs, requires a double-stranded RNA-binding protein partner to catalyze miRNA maturation. Does a single Dicer protein partner suffice for all pre-miRNA sequences and structures, or do different double-stranded RNA-binding proteins function as Dicer partners for distinct classes of pre- miRNAs? Some miRNAs reside in the 5' arm of the pre-miRNA stem; others, in the 3' arm. What sequence and thermodynamic features of the pre-miRNA ensure that the right miRNA is produced from the correct arm of the pre-miRNA stem? miRNAs function in protein-RNA complexes containing at their core a member of the Argonaute family of proteins. Flies have five different Argonaute proteins; humans have at least seven. How[unreadable]and why[unreadable]are miRNAs partitioned among different Argonaute proteins? What determines with which Argonaute protein a miRNA associates?Are complexes containing the same miRNA, but a different Argonaute protein, functionally distinct, each specialized for a different type of mRNA target? We seek to understand the biological functions of miRNAs in flies and humans. Why do flies only modestly impaired in miRNA production die young? Are miRNAs required for resistance to environmental stress? Finally, to provide a systems-level view of the miRNA pathway, we will develop new experimental tools to identify the mRNA species a miRNA regulates and through which Argonaute protein.